Ghrelin signalling in ß-cells regulates insulin secretion and blood glucose.

Insulin secretion from pancreatic islet ß-cells is stimulated by glucose. Glucose-induced insulin release is potentiated or suppressed by hormones and neural substances. Ghrelin, an acylated 28-amino acid peptide, was isolated from the stomach in 1999 as the endogenous ligand for the growth hormone (GH) secretagogue-receptor (GHS-R). Circulating ghrelin is produced predominantly in the stomach and to a lesser extent in the intestine, pancreas and brain. Ghrelin, initially identified as a potent stimulator of GH release and feeding, has been shown to suppress glucose-induced insulin release. This insulinostatic action is mediated by Gα(i2) subtype of GTP-binding proteins and delayed outward K⁺ (Kv) channels. Interestingly, ghrelin is produced in pancreatic islets. The ghrelin originating from islets restricts insulin release and thereby upwardly regulates the systemic glucose level. Furthermore, blockade or elimination of ghrelin enhances insulin release, which can ameliorate glucose intolerance in high-fat diet fed mice and ob/ob mice. This review focuses on the insulinostatic action of ghrelin, its signal transduction mechanisms in islet ß-cells, ghrelin's status as an islet hormone, physiological roles of ghrelin in regulating systemic insulin levels and glycaemia, and therapeutic potential of the ghrelin-GHS-R system as the target to treat type 2 diabetes.

Effect of fluoride ions on apatite crystal formation in rat hard tissues.

Fluoride is widely believed to be a useful chemical substance for preventing dental caries. However, the mechanism underlying crystal perforation in the tooth enamel and the effect of fluoride on hard tissues are unclear. To clarify the mechanism of the biological action of fluoride in the mineralization process, we examined the hard tissues of rats having received water containing a relatively low fluoride level. Electron microscopy revealed that fluoride ions could interrupt the crystal nucleation process, resulting in crystal perforation in the developing tooth enamel and the presence of amorphous minerals in bone crystals. Furthermore, the results of enzymatic analyses indicated that fluoride directly interfered with the synthesis of carbonic anhydrase by the enamel-forming cells, rather than being directly involved in the crystal formation. From the results, we would like to provide a possible mechanism of crystal perforation in the enamel induced by fluoride intake. Also, we would like to suggest that regardless of its amount, fluoride intake has harmful effects on both tooth and bone formation.

Receptor-operated regulation of ATP-sensitive K+ channels via membrane phospholipid metabolism.

ATP-sensitive K(+) channels (K(ATP)channels) regulate insulin secretion by coupling intracellular metabolic changes to excitability of the plasma membrane in pancreatic beta-cells. The channels are closed when extracellular glucose levels are elevated due to enhanced feature. By contrast, cardiac-type K(ATP) channels, which open in response to metabolic stress during cardiac ischemia, shorten action potential durations. This may contribute to the cardioprotection by decreasing Ca(2+) influx through sarcolemma. By sensing intracellular ATP levels or ATP/ADP ratios, changes in activity of K(ATP) channels convert metabolic information into membrane excitability. In addition to channel regulation by nucleotide concentrations, the channel activity is also dependent on the concentrations of membrane phospholipids, including phosphatidyl inositol-4,5-bisphosphate (PIP(2)). The levels of PIP(2) in the membrane may determine the basal activity of the channels. This suggests that channel activity would be modulated by the pathway of receptor-coupled GTP-binding protein (G-protein) and phosphatidyl inositol phospholipase C (PI-PLC) stimulation, which brings about depletion of the membrane PIP(2) pool. Thus, K(ATP) channels not only provide interface of metabolic changes with electrical excitation, but also rapidly transmit extracellular signals through receptor-coupled G-protein and PI-PLC pathway via PIP(2) metabolism.

High-resolution electron microscopy of the crystallites of fossil enamels obtained from various geological ages.

To elucidate the stability of the central dark line (CDL) in biologically induced hydroxyapatite crystals, we examined the diagenetic changes on the microstructures of the crystallites during the course of fossilization. Using transmission electron microscopy, we investigated the enamel crystallites of fossil animals of various geological ages ranging from Pleistocene to Cretaceous. Electron micrographs indicated that the microstructures and lattice images of each crystallite in fossil enamels were well-preserved regardless of the thickness of the enamel layer, and the presence of CDLs in fossil enamel crystals was also confirmed. The results indicated that the microstructure of hydroxyapatite crystals containing lattice images of CDLs appear stable during long geological periods. In addition, we conclude that the existence of lattice images in apatite with CDLs may be an indicator for the assessment of the evolution of dental enamel from fossil remains.

Free radical-mediated tolbutamide desensitization of K+ATP channels in rat pancreatic beta-cells.

To study the effects of hydroxyl radicals on the sensitivity of the ATP-sensitive K+ (K+ ATP) channel to tolbutamide, we used patch clamp and microfluorometric techniques in pancreatic beta-cells isolated from rats. cell-attached membrane patches, exposure of the cells to 0.3 mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8 mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8 microM before and immediately after exposure to H2O2. After prolonged exposure (>20 min) to H2O2, the IC50 was increased to 15 microM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1 mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10 microM tolbutamide. Addition of 0.3 mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10 min, followed by a sustained pronounced increase in [Ca2]i. After more than 20 min of exposure of cells to 0.3mM H2O2, [Ca2]i was increased to above 2 microM. Treatment of the cytoplasmic face of inside-out membrane patches with 1 microM Ca2+ attenuated the tolbutamide-sensitivity of the K+ATP channel, but not the ATP-sensitivity of the channel. These findings indicate that H2O2 reduces tolbutamide sensitivity by inducing a sustained increase in [Ca2+]i.

State-dependent modification of ATP-sensitive K+ channels by phosphatidylinositol 4,5-bisphosphate.

With inside-out patch recordings in ventricular myocytes from the hearts of guinea pigs, we studied ATP-sensitive K+ (K(ATP)) channels activated by phosphatidylinositol 4,5-bisphosphate (PIP2) with respect to sensitivity to ATP when in either a rundown state (RS) or a non-rundown state (NRS). Rundown of K(ATP) channels was induced by exposure either to ATP-free solution or to ATP-free solution containing 19 microM Ca2+. Exposure of membrane patches to 10 microM PIP2 reactivated channels with both types of rundown. The reactivation by PIP2 did not require ATP in the bath. The IC50 of channels recovered from RS and before the rundown was 37.1 and 31.1 microM, respectively. PIP2 irreversibly increased the mean current when the channel was in the NRS. This was associated with a shift of IC50 to 250.6 microM after PIP2 exposure. PIP2 activates NRS K(ATP) channels by decreasing their sensitivity to ATP, whereas PIP2 reactivates RS-K(ATP) channels independently of ATP without changing ATP sensitivity.

Demonstration of the central dark line in crystals of dental calculus.

Using an electron microscope and Fourier transform infrared (FTIR) microspectroscopy, we studied the lattice images of crystallites of dental calculus to demonstrate the presence of the central dark line (CDL) in its crystallite and to compare this CDL with that of bone and synthetic hydroxyapatite crystals. Ultrastructural observations revealed clearly a number of crystallites, which displayed a proper lattice image and CDL similar to that of bone, in the dental calculus. FTIR microspectroscopy revealed that the dental calculus displayed a set of major spectra analogous to that of bone. These results suggest that the formation process of hydroxyapatite crystals with CDL in dental calculus, which is considered to be an unusual type of calcified structure in association with microorganisms, is basically similar to that of the ordinary calcifying hard tissues (bone, enamel, etc.).

PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells.

The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells.

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.

Control of catecholamine release and blood pressure with octreotide in a patient with pheochromocytoma: a case report with in vitro studies.

A 65-year-old male patient with pheochromocytoma, whose hypertensive episodes were uncontrolled using conventional therapy, was successfully treated with octreotide (SMS 201-995). The serum catecholamine level and the urinary excretion of catecholamines decreased after 300 microgram/day of octreotide was administered. To clarify the mechanisms of octreotide that lower catecholamine released from a tumor, we studied the in vitro effects of octreotide on membrane potentials and voltage-dependent Ca(2+) channel (VDCC) current using the whole-cell patch-clamp technique in single pheochromocytoma cells dispersed after tumor resection. The action potentials were reversibly inhibited with 10 microM octreotide. In addition, the VDCC current evoked by depolarized pulses from the holding potential of -60 mV was inhibited with 10 microM octreotide. Octreotide is useful for controlling blood pressure before surgery in some patients with uncontrolled hypertension caused by a pheochromocytoma.

On the mechanism of ADP-induced alteration of sulphonylurea sensitivity in cardiac ATP-sensitive K(+) channels.

1. To study the mechanism of regulation of sulphonylurea sensitivity in ATP-sensitive K(+) (K(ATP)) channels, we used the inside-out patch clamp technique in guinea-pig ventricular myocytes. 2. In the absence of nucleotides, the half maximal concentration of tolbutamide inhibition of K(ATP) channels (IC(50)) was 0.4 mM, and it decreased to 0.1 mM when 0.1 mM ATP was added. 3. Increasing the ADP concentration from 0 to 0.1 and 0.3 mM in the absence of ATP shifted the IC(50) from 0.4 to 5.3 and 11.4 mM, respectively. Increasing the ADP concentration further to 1 and 3 mM conversely reduced the IC(50) to 9.5 and 4.4 mM, respectively. 4. In the absence of Mg(2+) and ADP, the IC(50) was calculated to 16.6 mM which was found to be less, 12.3, 5.1 and 2.5 mM, respectively, when the ADP concentration was increased to 0.1, 0.3 and 1 mM. 5. The IC(50)s for tolbutamide obtained at various concentrations of ADP in the presence of Mg(2+) were best fitted by equations reflecting a model that assumed two binding sites for ADP; one is a high affinity site that reduces the sensitivity to the sulphonylurea, while the other is a low affinity site that increases such sensitivity. Dissociation constants calculated for ADP to sites 1 and 2 were 2.6 microM and 46.7 mM, respectively. In the absence of Mg(2+), data were fitted by equations corresponding to a single site model (site 2); the dissociation constant for ADP was 25.0 mM. 6. It is concluded that ADP modifies tolbutamide sensitivity by binding to two sites. The high affinity site is strongly Mg(2+)-dependent, whereas the low affinity site is Mg(2+)-independent.

P2Y-purinoceptor mediated inhibition of L-type Ca2+ channels in rat pancreatic beta-cells.

We used the patch-clamp technique to study the effects of extracellular ATP on the activity of ion channels recorded in rat pancreatic beta-cells. In cell-attached membrane patches, action currents induced by 8.3 mM glucose were inhibited by 0.1 mM ATP, 0.1 mM ADP or 15 microM ADPbetaS but not by 0.1 mM AMP or 0.1 mM adenosine. In perforated membrane patches, action potentials were measured in current clamp, induced by 8.3 mM glucose, and were also inhibited by 0.1 mM ATP with a modest hyperpolarization to -43 mV. In whole-cell clamp experiments, ATP dose-dependently decreased the amplitudes of L-type Ca2+ channel currents (ICa) to 56.7+/-4.0% (p<0.001) of the control, but did not influence ATP-sensitive K+ channel currents observed in the presence of 0.1 mM ATP and 0.1 mM ADP in the pipette. Agonists of P2Y purinoceptors, 2-methylthio ATP (0.1 mM) or ADPbetaS (15 microM) mimicked the inhibitory effect of ATP on ICa, but PPADS (0.1 mM) and suramin (0.2 mM), antagonists of P2 purinoceptors, counteracted this effect. When we used 0.1 mM GTPgammaS in the pipette solution, ATP irreversibly reduced ICa to 58.4+/-6.6% of the control (p<0.001). In contrast, no inhibitory effect of ATP was observed when 0.2 mM GDPbetaS was used in the pipette solution. The use of either 20 mM BAPTA instead of 10 mM EGTA, or 0.1 mM compound 48/80, a blocker of phospholipase C (PLC), in the pipette solution abolished the inhibitory effect of ATP on ICa, but 1 microM staurosporine, a blocker of protein kinase C (PKC), did not. When the beta-cells were pretreated with 0.4 microM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, ATP lost the inhibitory effect on ICa. These results suggest that extracellular ATP inhibits action potentials by Ca2+-induced ICa inhibition in which an increase in cytosolic Ca2+ released from thapsigargin-sensitive store sites was brought about by a P2Y purinoceptor-coupled G-protein, PI-PLC and IP3 pathway.

A case of renal juxtaglomerular cell tumor: usefulness of segmental sampling to prove autonomic secretion of the tumor.

A 27-year-old female patient had been treated for hypertension with conventional therapy for years, because renal vein renin levels failed to show lateralization in renal venous samplings and a renal juxtaglomerular cell tumor (RJGCT) had gone undiagnosed. Abdominal computed tomography revealed a mass at the middle of the right kidney. The right renal venogram demonstrated distinct segmental veins from the upper pole and from the middle and lower poles in the right kidney. On segmental renin sampling from each renal vein, the plasma renin concentration (PRC) of the segmental veins from the middle and lower poles was higher than that from other sites. We diagnosed RJGCT of the right kidney and performed right-sided nephrectomy. After the resection, the PRC rapidly decreased. Immunohistochemical studies using antihuman renin antibodies revealed positive staining of the tumor cells. It is an important strategy to make a segmental sampling at the site as close as possible to the RJGCT.

Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L-type Ca2+ channels in rat islet beta-cells.

1. Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG-CoA reductase inhibitors. Alteration of pancreatic beta-cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG-CoA reductase inhibitors on beta-cell function. 2. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of beta-cell function. The present study examined the effects of HMG-CoA reductase inhibitors on the glucose-induced [Ca2+]i signalling and insulin secretion in rat islet beta-cells. 3. Simvastatin, a lipophilic HMG-CoA reductase inhibitor, at 0.1-3 microg ml(-1) concentration-dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3 mM glucose in single beta-cells. The less lipophilic inhibitor, simvastatin-acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100 microg ml(-1), was without effect on [Ca2+]i. 4. Simvastatin (0.3 microg ml(-1)), more potently than simvastatin-acid (30 microg ml(-1)), inhibited glucose-induced insulin secretion from islets, whereas pravastatin (100 microg ml(-1)) had no effect. 5. Whole-cell patch clamp recordings demonstrated a reversible inhibition of the beta-cell L-type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L-arginine and KCl, agents that act via opening of L-type Ca2+ channels. 6. In conclusion, lipophilic HMG-CoA reductase inhibitors can inhibit glucose-induced [Ca2+]i signalling and insulin secretion by blocking L-type Ca2+ channels in beta-cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG-CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.

Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.

1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (KATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2DeltaC26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 microM Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1 % (mean +/- s.e.m.) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2 % (n = 13) of the Ca2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2 % (10 microM) and 4.3 +/- 2.5 % (100 microM) of the Ca2+-induced run-down. 4. MgUDP (10 mM) reversed the Ca2+-induced run-down of Kir6.2/SUR2A channels by 60.4 +/- 7.6 % (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2DeltaC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca2+; Kir6.2DeltaC26 channels were inactivated to 44.8 +/- 4.4 % (n = 14) by 100 microM Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2DeltaC26 by 89.8 +/- 7. 7 % (n = 9), and 100 microM wortmannin inhibited this reaction (1.8 +/- 2 %, n = 7). 6. Application of 10 microM phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4 %, n = 10). Even after the MgATP-dependent recovery was blocked by 100 microM wortmannin, PI-4,5-P2 reactivated the channels (102.3 +/- 8.6 %, n = 5). Similar results were obtained with Kir6.2DeltaC26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P2 or phosphatidylinositol-3,4, 5-trisphosphate may upregulate Kir6.2 channels.

Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6.

We examined the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial matrix Ca2+ concentration ([Ca2+]m) in the pancreatic beta-cell line, MIN6. [Ca2+]c was monitored in a single or a group (30 cells) of fura-2-loaded MIN6 cells, and [Ca2+]m was measured in a group (1 x 10[6] cells) of MIN6 cells stably transfected with aequorin targeted at the mitochondria. Exogenous ATP (0.25 mmol/l) produced a single transient increase in [Ca2+]c whereas 22 mmol/l KCl produced a sustained plateau increase. ATP and KCl evoked transient increases in [Ca2+]m but with distinct time courses of [Ca2+]m decline: the [Ca2+]m increase induced by ATP decreased more rapidly than that induced by KCl. Nitrendipine (3 micromol/l), a blocker of L-type Ca2+ channels, inhibited both [Ca2+]c and [Ca2+]m signals in response to KCl and tolbutamide, but not those to ATP. Peak levels of [Ca2+]m increase (around 2 micromol/ l) exceeded those of [Ca2+]c increase (around 500 nmol/l). A rise in glucose concentration from 3 to 30 mmol/l induced oscillations of [Ca2+]c that overlay the sustained increases in [Ca2+]c in single cells. An oscillatory increase in [Ca2+]m was similarly observed in response to glucose. Addition of 10 mmol/l 2-ketoisocaproic acid at 20 mmol/l glucose further increased the plateau level of [Ca2+]c and the frequency of [Ca2+]c oscillations, which were correlated with a further increase in [Ca2+]m. In response to pulsatile exposure to KCl, [Ca2+]c and [Ca2+]m increased synchronously. These data suggest that an oscillatory increase in [Ca2+]m in beta cells, the signal which is thought to be necessary for continuous stimulation of mitochondrial metabolism, is produced synchronously with the [Ca2+]c oscillations.

Behavior of carbonate and magnesium ions in the initial crystallites at the early developmental stages of the rat calvaria.

Analysis of the contents of calcium (Ca), magnesium (Mg), phosphate, and carbonate ions in the mineral phase of rat calvaria specimens obtained at different developmental stages indicated that the mineral at the newborn stage contained a negligible amount of carbonate, but a high content of Mg. There was no significant difference in Ca and phosphate (as PO4) contents between the newborn material and that from later stages. A relatively large amount of carbonate was detected in the calvaria from 6-day-old rats, in which only immature crystals were observed, thus indicating the beginning of apatite formation. Furthermore, using laser Raman microprobe analysis we confirmed that the Raman peak at 1120 cm-1 band, indicative of a Mg-CO3 compound, appeared at the 6-day stage. We also observed that the Raman peak at 988 cm-1 found in the samples from the newborn seemed to have shifted to 963-962 cm-1 in the case of those obtained from 6-day-old rats, a shift which suggests the conversion from the non-apatitic to the apatitic form. These results indicate that carbonate ions might facilitate the initiation of crystal development by converting the inhibitory Mg ion into its inactive form (Mg-carbonate compound).

Pure ovarian ependymoma: report of a case treated with surgery, chemotherapy, irradiation and hyperthermotherapy.

Ependymomas usually develop from neuroectodermal organs. Pure ovarian ependymoma is an extremely rare tumor. We report a patient with ovarian ependymoma who died at the age of 28, 9 years after initial surgery and subsequent intensive combination therapy (chemotherapy, irradiation and hyperthermotherapy) for repeated relapses and metastatic tumors. The diagnosis was confirmed by histopathological and immunohistochemical studies. For recurrent and persistent ependymoma, a combination of the treatment modalities described above is suggested to be beneficial in attenuating the rapid progress and spread of this disease.